Specific bifunctional BY-001 (active composition of homomultimer of chimeric protein PD-L1 / Fc-gamma1) down regulates the activation of human immune cells and the use thereof

ABSTRACT

The present invention provides an active form (BY-001) of a homomultimeric chimeric protein PD-L1/Fc-gamma1, which may have a composition of 25% dimer, 30% tetramer and 45% hexamer. BY-001 consists of two distinct functional domains at the aggregated state, one is the extracellular domain of PD-L1 and another one is the IgG1 Fc region. Accordingly, the first functional domain of PD-L1 at its aggregated state is more effective in suppressing the activity of CD3+ and CD28+ T cells; while, on the other hand, the second functional domain of FC-gamma1 at its aggregated state exerts only the suppressive effects on the immune cell bearing the Fc-gamma receptors. Thus, the features provided in present invention indicate that BY-001 regulates the functions of immune cells subsets that are responsible for the peripheral immune tolerance, which makes BY-001 potential to help restore or maintain the peripheral immune tolerance in patients with autoimmune diseases or in recipients of allografts.

1. BACKGROUND OF THE INVENTION

The immune system normally recognizes and defends against foreignpathogens but does not respond to self-components of tissues, suchintrinsic state is established and maintained precisely by the processof immune tolerance. The inflammatory damage of one or more organsystems caused by inappropriate activation of self-reactive both oreither T cells and B cells is due to the loss of balance betweeneffectors (B cells and T cells) [1, 2] and regulatory components (Tregcells and dendritic cells) of the immune system [3]. Therefore,restoration of immune tolerant state is the ultimate goal in thetreatment of autoimmune diseases [4, 5].

Cell surface molecules such as CTLA-4 (cytotoxic T-lymphocyte-associatedprotein 4, or CD152) and PD-1 (Programmed Death-1, also known as CD279)deliver inhibitory immunoregulatory signals that are thought to becrucial to the maintenance of normal immune tolerance [6, 7] through itsrecognizing and binding to CD80 (B-7.1) or CD86 (B-7.2). Based on thediscoveries, Bristol-Myers Squibb, a pharmaceutical company, developed achimeric protein composed of the Fc region of the immunoglobulin IgG1and the extracellular domain of CTLA-4 named as Abatacept (CTLA-4/Fc),thereafter, it was approved for clinic application by FDA in 2011.However, the pharmacological mechanism of Abatacept is to serve asantagonist of CD28 to interrupt the second signal, CD28 binding to B-7for TCR activation, instead of the delivery of checkpoint signal throughCTLA-4. Therefore, Abatacept interrupts the systemic cellular immuneresponse, which, as a side effect, may cause serious infectiousdiseases, even cancer.

Second cell surface molecule responsible for immune checkpoint is PD-1(Programmed cell death protein 1, also known as CD279), which isstrictly expressed on thymocytes, activated T cells and pro-B cells. Ithas been believed that PD-1 is related biasedly to the maintenance ofperipheral immune tolerance when it is associated with either of its twoidentified ligands: 1), Programmed Death-1 ligand 1 (PD-L1), also knownas CD274 or B7-H1, broadly expressed on the hematopoietic andnonhematopoietic cells including tumor cells; 2), Programmed Death-1ligand 2 (PD-L2), also known as CD273, or B7-DC strictly expressed onthe dendric cells and macrophages. The PD-1 engagement with PD-Lsresults in suppressing the activation of T cell or differentiation ofmature B cell. Thus far three mechanisms have been elaborated for thepivotal role of PD-1/PD-L1 pathway in immune tolerance: (1) Induction ofT cell anergy [8] through PD-L1 binding to PD-1 then activating the SHP2to suppress the TCR/CD28 signaling; (2) suppression of B cell maturationthrough PD-L1 binding to PD-1 expressed on pre-B cells then activatingthe SHP2 to suppress the BCR signaling; [9]; (3) generation ofregulatory T cells (especially type 1 (Tr1)) [10] and conversion ofhuman TH1 into Treg cell (iTreg cell) [11]. In addition, soluble formsof PD-1 (sPD-1) and PD-L1 (sPD-L1) in peripheral blood may also be asource for the regulatiOn of PD-1/PD-L1 pathway in immunity [12, 13]. Aperfect manifestation of such mechanisms is that certain tumor cells usePD-L1 overexpression on their cell membranes and simultaneously shedinto the circulation to successfully escape immune attacks [14].

In contrary with in tumor, according to the latest findings inautoimmune diseases, the weakening or deletion of the PD-1/PD-L1 pathwayin immune cells is directly related to the instability or destruction ofperipheral immune tolerance [10]. Mounting evidence demonstrate thatimpaired PD-1/PD-L1 function plays an important role in a variety ofautoimmune diseases, such as SLE (systemic lupus erythematosus) and RA(Rheumatoid Arthritis), etc. [15]. The animal model demonstrates thatloss of signaling of PD-1/PD-L1 (PD-1 knockout mice) develops lupus-likeautoimmune disease [16], or autoimmune dilated cardiomyopathy dependingupon the genetic background [17]. PD-L1 deficiency enhances diseaseprogression in the experimental autoimmune encephalomyelitis [18],nonobese diabetic (NOD) model of autoimmune diabetes [19, 20]and themurine model of multiple sclerosis (MS)[21]. Tissue expression of PD-L1mediates peripheral T cell tolerance [22].

The third cell surface molecule responsible for immune tolerance isFc-gamma receptor type IIB (one of Fc-gammaRIIA/B, collectively known asCD32). Human Fc-gamma receptors have been identified so far arehFc-gammaRI (CD64), hFc-gammaRll (types A and B, collectively known asCD32) and hFc-gammaRIII (types A and B, collectively known as CD16).Each type of receptor exhibits distinctive tissue distribution,structure and binding specificity towards various IgG subclasses (7,8).Fc-gammaRIIB is restrict expressed on B cell, monocyte/macrophage anddendritic cell in human, while basophil, eosinophil and mast cell aswell in mice. Fc-gammaRII displays low affinity for monomeric IgG buthigh-avidity for aggregated multimeric IgG, which are particularlyimportant in the recognition and binding of antibody—antigen complexesduring an immune response [23]. The B cell stage(s) at whichFc-gammaRllB exerts its function as a gatekeeper of self-tolerance hasrecently been defined [24, 25]. The main function of Fc-gammaRIIB is toinhibit activating signals, which is achieved through co-ligation ofFc-gammaRIIB with either activating Fc-gammaRs or with the BCR by immunecomplexes ([26]). This leads to phosphorylation of the cytoplasmicdomain ITIM of Fc-gammaRllB by the Src-family kinase IYN. Thisphosphorylation event is thought to require access of Fc-gammaRIIB tosphingolipid rafts in which activating Fc-gammaRs and the BCR residefollowing cross-linking. Subsequent binding of SH2-domain-containinginositol phosphatases (SHIPs), in particular SHIP1, result in thedephosphorization of downstream targets and inhibition of the activatingsignaling cascade. There is accumulating evidence that Fc-gammaRllBmediates its function during late stages of B cell maturation, thusrepresenting a distal checkpoint [25]. Through the analysis of ananti-DNA knock-in model, it was established that the absence ofFc-gammaRIIB resulted in the expansion of IgG-positive plasma cellssecreting autoreactive antibodies (Fukuyama et al., 2005). Here,Fc-gammaRIIIB might serve as the final barrier to prevent these B cellswith potentially harmful BCR specificities from maturing into plasmacells that would otherwise induce tissue pathology by secretion of largeamounts of self-reactive antibodies. Another cell type whereFc-gamagRIIB may play an important role in regulating immunity andtolerance are the dendritic cells [27-29]. A number of chronicinflammatory diseases have been shown to be associated with Fc-gammareceptor genetic variants and include (but are not limited to)autoimmune pathologies, such as systemic lupus erythematosus (SLE)[30],rheumatoid arthritis[31], acute allograft rejection [32] and vascularinflammatory and thrombotic disorders, such as coronary artery stenosis,peripheral atherosclerosis and vasculitis [33, 34].

Since PD-1 is expressed in activated T cells and B cells, it is expectedthat the PD-L1 protein can be effectively used as a therapeutic agentthat specifically targets activated immune cells to suppress theinflammatory reaction not only in an autoimmune disease but also inorgan transplantation. However, Fc-gammaRIIB is expressed in dendriticcells and monocytes/macrophages, it is expected also that aggregatedform of Fc-gammal can be used as a therapeutic agent that specificallytargets these immune cells to suppress the inflammatory reaction duringthe autoimmunity. Because the induction and maintenance of peripheralimmune tolerance are dependent on the orchestra playing by all type ofthose cell populations. To date a peripheral immune tolerance-basedimmunotherapeutic agent using an agonist to rebuild the tolerancethrough both pathways of PD-1/PD-L1 and Fc-gamma/Fc-gamma receptors hasnot yet been developed. Therefore, there is a need to develop atherapeutic reagent that the therapeutic effect not only targeting theautoreactive T cells and B cells but also targeting the inappropriatelyactivated monocytes/macrophages and dendritic cells in inflammatorytissues, meanwhile, such reagent will avoid the causes of antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC) in vivo due to its feature of biding to theactivation of Fc receptor of Fc-gammaRI. Thus, when Ig chimeric proteinsare used as an agent for treating an autoimmune disease or as an agentfor inducing immune tolerance in organ transplantation, they cannot playthe role of inhibiting inflammatory responses, and may rather aggravateinflammation.

2. SUMMARY OF THE INVENTION

In first aspect, the invention provides a bispecific biologic (chimericprotein of PD-L1/FC-gamma1) named BY-001, which is a composition ofhomomultimeric forms of dimer, tetramer and Hexamer, wherein the ratioof the forms is at 25% of dimer, 30% of tetramer and 45% of Hexamerrespectively.

In second aspect, current invention provides the demonstrations ofbispecific function of BY-001 that aggregated PD-L1 can trigger thepathway of PD-1 to decrease the cytokine production from activated Tcell line (jurkate cell expressing PD-1), and also the aggregatedFc-gamma1 shows the suppressive effect on monocyte/macrophage cell line(THP-1 cell expressing Fc-gamma1RI & II) and erythroid cell line (K562cell expressing Fc-gamma1RII).

In third aspect, the present invention also demonstrates that thefunctional features of both the domains PD-L1 and Fc-gamma1 in BY-001fully inherit their characteristics in their parental proteins that havebeen confirmed in vitro and in vivo that published in highly recognizedscientific journals. Therefore, an obvious perspective emerges that theBY-001 of the present invention is potential for a pharmaceuticalcomposition for treating an autoimmune disease caused by abnormalregulation of an immune response or preventing the rejection ofallografts.

3. DISCLOSURE 3.1. Technical Problem and Solution

An object of the present invention is to provide BY-001 (a compositionof bifunctional homomultimeric forms chimeric protein PD-L1/Fc-gamma1)comprises the forms of dimer, tetramer and Hexamer at the ratio of 25%,30% and 45%, wherein monomer form of chimeric protein PD-L1/Fc-gammalconsists of two distinct domains. The amino acids of cysteine in theregion of immunoglobulin Fc-gammal are kept in order to form themultimeric PD-L1/Fc-gamma1, such homomultimeric form provides theaggregated Fc-gamma1 (BY-001) for binding to Fc-gammaRIIB instead ofFc-gammaRI. Another object of the present invention is to provide theaggregated form (BY-001) of extracellular domain of PD-L1 protein, whichshows an excellent regulating effect on activity of T cells. Thirdobject of the present invention is to provide the aggregated form ofFc-gamma1 region, which binds only to Fc-gamma1RIIA and B and suppressesthe production of pro-inflammatory factors in monocyte/macrophages andpre-erythrocytes.

3.1. Advantageous Effects

BY-001 of the present invention is a composition of chimeric proteinPD-L1/Fc-gamma1 in the forms of dimer, tetramer and Hexamer. Suchaggregated state of the chimeric protein PD-L1/Fc-gamma1 displays higherregulatory efficacy to PD1 and Fc-gammaRII. Therefore, it is morepotential to regulate the activities of T cells including Th cells,cytotoxic-T cells and B cells during the active stage of autoimmunity,as well as to regulate the activities of monocytes/macrophages,dendritic cells and iTreg cells to restore and maintain the peripheralimmune tolerance. Thereafter, the BY-001 of the present invention ispotential for a pharmaceutical composition for treating an autoimmunedisease caused by abnormal regulation of an immune response orpreventing the rejection of allografts.

4. DESCRIPTION OF DRAWING

FIG. 1. Design for Chimeric Peptide of PD-1L/Fc-gammal (BY-001) and ItsAmino Acid Sequence A. Schematic diagram for the design of chimericprotein PD-L1/Fc-gmma1 consisting of extracellular portion of humanPD-L1 and constant region of human IgG1 Fc. Please be noted:

-   -   1). Cysteines in V-set, C-set and IgG1Fc regions are kept for        the —S—S— “disulfide” bond to form the advanced structure forms.    -   2). PD-L1/FC1 is 449 amino acid in total    -   3). The signal peptide of PD-L1 comprises amino acids 1-18,        which will be digested when the protein secrets into culture        medium. Therefore, the purified PD-L1/Fc-gamma1 of current        invention contains 431 amino acids in total.    -   4). immunoglobulin V like region (V-set, 19˜127 amino-acids,        exon-2)    -   5). immunoglobulin constant like region (C-set, 133˜220        amino-acids, exon 3)    -   6). human IgG1 heavy chain constant domain (C region, 221⁻449)        is the region of Fc-gamma1.

B. The AA sequence of PD-L1 contained in chimeric proteinPD-L1/Fc-gammal. The sequence highlighted in yellow is the part ofsignal sequence for leading the protein toward the secretory pathway,which is cut away as the protein released to medium. The sequencehighlighted in shadow is the region of Fc-gamma1. For the detail aboutsequences, please refer to the information of file“BY-001sequence_ST25”.

FIG. 2. Visualized Protein of BY-001 in SDS-Page Gel and Western-BlotDNA fragment encoding amino acid sequence of PD-L1 stated in (FIG. 1.B)was inserted into vector of pFUSE-hIgG1-Fc-gamma1purchased fromINVIVOGEN USA, 10515 Vista Sorrento Pkwy, San Diego, Calif. 92121, USA.The DNA of pFuse-PD-L1/Fc-gamma1 was transfected with 293 cells.Monofinity A Column Purification (GenScript, 860 Centennial Avenue,Piscataway, N.J. 08854) was utilized to purify the BY-001 from thesupernatants of 293 cell culture. The molecular wieght of expectedprotein in monomeric form is about 60 kilodalton (KD). Westen blotsystem was used for the analysis of protein product. Reducing andnonreducing condition (without dithiothreitol (DTT) for the former, withDTT for the latter) were applied for breaking up the disulfide bounds inadvanced structure of BY-001.

A. SDS-page gel, Coommassie blue staining. Lane M1: Protein Marker Cat.No. 3452, Lane 1: BY-001 at Reducing condition (monomer), Lane 2: BY-001at non-reducing condition (no monomer, but dimer, tetramer and Hexamer).

B. Western blot probed with Primary antibody: Goat Anti-Human IgG-HRP.Lane M2: Protein Marker, GenScript, Cat. No. M00521, Lane P: Human IgG1,Kappa (Sigma, Cat.No. 15154) as a positive control. Lane 1, monomer ofPD-l1/Fc-gamma1 band at nonreducing condition; Lane 2, multimeric formsof BY-001, dimer, tetramer and hexamer.

C. Western blot probed with Human PD-L1/B7-H1 Antibody (R&D,Cat.No.AF156). Lane M2: Protein Marker, GenScript, Cat. No. M00521; Lane1, monomer of PD-l1/Fc-gamma1 band at nonreducing condition; Lane 2,multimeric forms of BY-001, dimer, tetramer and hexamer.

D. Diagrams of the BY-001, cartoons of D, E and F are the graphic viewfor illustration of BY-001 at forms of dimer, tetramer and hexamer.

FIG. 3. BY-001 suppresses IL-2 production from Jurkate cell line Jurkate(from ATCC, USA) is a T cell line generated from human T-cell leukemia.CD3, CD28 and PD-1 are molecules expressed on the cell surface. OKT3 isan antibody against CD3, and its binding and cross-linking will activateT cells. CD28 is a receptor for co-stimulatory molecule B7 family. Theassociation of anti-CD28 with CD28 will greatly enhance T cellactivation based on OKT3 stimulation in vitro. IL-2 production is one ofthe biomarkers commonly to indicate T cell activation. The goal of thisexperiment is to validate and demonstrate the function of BY-001 inregulation of T cell activation in vitro. 5ug/ml of OKT3 (Anti-humanCD3, Clone OKT3, BD bioscience) was coated onto 96-well, U bottommicro-plate for overnight at 4° C. Cell suspension of Jurkate cells at 3million/ml is in RPM1164 medium with 10% FBS.

A. 100 ul of Cell suspension jurkate at 3 million/ml was added into eachwell hereafter, BY-001 was added into wells according to designed. Theplate was kept in incubator at 37° C. and 5% CO₂ overnight. The culturesupernatants were harvested from wells for the measurement of IL-2production. Human IL-2 High Sensitivity ELISA Kit (eBioscience, 1030Vienna, Austria) was used for the measurement of IL-2. The resultsindicate that BY-001 inhibits the IL-2 production effectively at thestarting concentration (0.1 ug/ml) of BY-001. CD3 signal along (withoutthe CD28 second signal) the inhibition was 87.5% at concentration of 0.1ug/ml of BY-001, such inhibition was persistent from the concentrationsof 0.1 ug/ml to 1 ug/ml.

B. Anti-human CD28 (BD bioscience, Clone CD28.2) mixed at 2 ug/ml intosame jurkate cell suspension solution as used in (A). The plate wasincubated at 37° C. and 5% CO₂ for overnight. The culture supernatantswere harvested from wells for the measurement of IL-2 production byfollowing the method in (A). The results indicate that TCR signal withthe help of co-stimulator signal make Jurkate cell produces higher levelof IL-2 (from 30 pg/ml at OKT3 along to 60 pg/ml at OKT3 + anti-CD28),however it makes BY-001 inhibit IL-2 production as a gradient decentpattern at the concentration (0.5 to 2 ug/ml), the inhibitory ratebecome to be 13.1% at 0.5ug/ml, 43% at 1.0ug/ml to 67.6% at 2ug/ml ofBY-001.

FIG. 4. BY-001 suppresses IL-2 production from PBMC human primary cellsThe PBMC was obtained from healthy human donor. PBMC are cellscontaining only mononuclear in their cell. Among the PBMC, CD3 isexpressed on all T cells, while CD3 and CD28 double positive T cellsaccount for the majority of all T cells. B7 family and PD-1 areexpressed on most of the PBMC cell surface. Fc receptor is expressed ondendritic cell, monocyte, etc. The goal of this experiment is tovalidate and demonstrate that BY-001 functions as a mediator inregulation of T cell activation in human primary cell, by which toindicate the great opportunity of success in clinical trial.

The 96-well plate was coated with 3 ug/ml of OKT3 at 4° C. forovernight. 100 ul of Jurkate cell solution at 3 million/ml in thepresence or no presence of Anti-human CD28 at 2 ug/ml were added intowells. The plate was kept in incubator at 37° C. and 5% CO₂ forovernight, supernatants were collected from the wells for IL-2 detectionwith Human IL-2 High Sensitivity ELISA Kit.

A. CD3 signal along (without the CD28 second signal) the inhibition was90.8% at concentration of 0.25 ug/ml of BY-001, the inhibition ispersistent at concentrations of BY-001 from 0.25 to 4 ug/ml.

B. CD3 signal together with the CD28 second signal the inhibitionpattern became to be gradient decent from the inhibitory rate 17.5% at0.25 ug/ml to 74.1% at 4 ug/ml of BY-001.

FIG. 5 BY-001 regulates BY-001 regulates IL-4 and TGF-betta productionfrom K562 cell K562 cell (from ATCC, USA) is pre-erythrocyte generatedfrom human myelogenous leukemia (of the erythroleukemia type) in whichno CD3, CD28 and PD-1 molecules are expressed. However, it was reportedthat only Fc-gamma1 receptor II are expressed on its cell surface but noFc-gamma1 receptor I. Therefore, only aggregated Fc-gamma1 are capableto binding to and triggering the Fc-gamma1/Fc-Gamma1RII pathway. Todetermine the function of aggreged Fc-gamma1 of BY-001 we measured theregulation of IL-4 and TGF-betta production in K562 cells. Apparently,the signaling transferred in this demonstration is suppressive.

A. The basal level of IL-4 production in K562 is high (84 pg/ml).Addition of BY-00-1 according to designed into the cells culturedecreases the IL-4 level. The inhibition is sufficient at concentrationof 4 ug/ml of BY-001, which indicates that aggregated Fc-gamma1 bind toFc-gamma1/Fc-Gamma1RII and trigger the inhibitory pathway.

B. The basal level of TGF-betta production in K562 is high (1250 pg/ml).Addition of BY-00-1 according to designed into the cells culturedecreases the TGF-betta level. The inhibition is sufficient atconcentration of 2 and 4 ug/ml of BY-001, which indicates thataggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger theinhibitory pathway.

C. The diagram for BY-001 binding to and triggering the Fc-gammaRII,thereafter to deliver the suppressive signal. ITAM stands forimmunoreceptor tyrosine-based activation motif in which the tyrosineresidues are phosphorylated upon the binding between Fc-gamma1 andFc-gamma1RII. While ITIM stands for the immunoreceptor tyrosine-basedinhibitory motif in which the tyrosine residues are phosphorylated,thereafter the phosphatase SHIP1 or 2 is activated to dephosphorylatethe phosphorylated tyrosine in ITAM of Fc-gammaRIIA or C via thecrosslinking of bound Fc-gamma1s of BY-001.

FIG. 6. BY-001 regulates the IL-4 and TGF-betta production from THP-1cells THP-1 cell (from ATCC, USA) is THP-1 is a human monocytic cellline derived from acute monocytic leukemia in which no CD3, CD28 andPD-1 molecules are expressed. However, it was reported that bothFc-gamma1 receptor I and II are expressed on its cell surface. To date,it has been believed that Fc region of monomeric antibody is highaffinity to Fc-gamma1RI but with very low avidity to Fc-gammaRII. Incontrast, the Fc portion of antigen-antibody complex binds toFc-gammaRII instead of Fc-gammaRI. Therefore, to determine the efficacyof aggregated Fc-gammal of BY-001 in binding to and triggering theFc-gamma1/Fc-gamma1RII pathway, we measured the regulation of IL-4 andTGF-betta production in THP-1 cells. Apparently, the signalingtransferred in this demonstration is suppressive, which indicates alsothat the aggregated Fc-gammal of BY-001 triggers the inhibitory pathwayof Fc-gamma/Fc-gammaRIIB.

A. The basal level of IL-4 production in THP-1 is high (86 pg/ml).Addition of BY-00-1 according to designed into the cells culturedecreases the IL-4 level. The inhibitions are sufficient atconcentration of 2 and 4 ug/ml of BY-001, which indicates thataggregated Fc-gammal bind to Fc-gamma1/Fc-Gamma1RII and trigger theinhibitory pathway only.

B. The basal level of TGF-betta production in THP-1 is high (485 pg/ml).Addition of BY-00-1 according to designed into the cells culturedecreases the TGF-betta level. The inhibition is observed atconcentration of 2 and 4 ug/ml of BY-001, which indicates thataggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger theinhibitory pathway only.

C. The diagram for BY-001 binding to and triggering the Fc-gammaRII,thereafter to deliver the suppressive signal. ITAM stands forimmunoreceptor tyrosine-based activation motif in which the tyrosineresidues are phosphorylated upon the binding between Fc-gamma1 andFc-gamma1RII. While ITIM stands for the immunoreceptor tyrosine-basedinhibitory motif in which the tyrosine residues are phosphorylated,thereafter the phosphatase SHIP1 or 2 is activated to dephosphorylatethe phosphorylated tyrosine in ITAM of Fc-gammaRIIA or C via thecrosslinking of bound Fc-gamma1s of BY-001. Apparently, the Fc-gammaRIhas no effect on the inhibition, which is benefited for BY-001,hereinafter, in dealing with autoimmunity.

FIG. 7 Advanced structural difference between BY-001 andantigen-antibody complex specific antibodies associate with antigen withelectrostatic attraction and hydrophobic bonds or hydrogen bonds, whichare separated easily in SDS page gel of protein electrophoresis. Whilethe PD-L1/Fc-gamma1 of present invention bound together with disulfidebound (S—S) without the component of antigen, and the S—S bound only canbe broken up under the of reducing condition (by adding DTT) in SDS pagegel of protein electrophoresis.

A. The diagram is the dimer of BY-001 bound together by S—S bound

B. The diagram is the tetramer of BY-001 bound together by S—S bound

C. the diagram is the hexamer of BY-001 bound together by S—S bound

D. the diagram is the antigen-antibody complex

FIG. 8 Expected mechanism for BY-001 to rebuild the peripheral immunetolerance Genetic alterations (gene mutation or polymorphism) may causethe individual difficult to maintain the peripheral tolerance ofimmunity. The dendritic cells, macrophages, T cells are become to bevery fragile to resist the alteration of microenvironment, then easilyto be activated inappropriately. The outcome is the generation ofauto-cytotoxic T cell against self-tissues. The addition of BY-001 willdirectly reduce the activity of cytotoxic T cells by which to induce theauto-cytotoxic T cell anergy, on the other hand, BY-001 will alsoregulate the activities of Th cell, Treg cells and dendritic cells,macrophages which is critical to rebuilt peripheral immune tolerance.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Definitions: Prior to describing the invention in more detail thefollowing definitions are provided. Unless otherwise stated, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Any methods and materials with similar or equivalentfunction to those described herein can be used in the practice ortesting of the present invention. Methods, devices, and materialssuitable for such uses are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention.

In accordance with the object of the present invention, in one aspect,there is provided a fusion protein comprising the extracellular domainof PD-L1 protein or a fragment thereof and a human immunoglobulinFc-gamma1 region (“PD-L1/Fc-gamma1 chimeric protein,” “PD-L1/Fc-gamma1protein,” “PD-L1 chimeric protein” or “chimeric protein”). Theextracellular domain of PD-L1 may be a polypeptide comprising animmunoglobulin V (V-set) like domain of PD-L1 and an immunoglobulin C(C-set) like domain of PD-L1. The extracellular domain of PD-L1 is adomain exposed outside a cell membrane, and may be a polypeptidecomprising the amino acids at positions 19 to 220 of sequence of FIG. 1B of current invention. In addition, the extracellular domain of PD-L1or a fragment thereof may be of human origin. In addition, theextracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to thepolypeptide sequence consisting of the amino acids at positions 19 to220 of sequence of FIG. 1B. In addition, Human IgG1 heavy chain constantdomain (C region, 221˜449) of the sequence of FIG. 1B is the region ofFc-gamma1. All the Cysteines in V-set and C-set and human immunoglobulinFc-gamma1 region of PD-L1/Fc-gamma1 thereof are kept in currentinvention for the —S—S— “disulfide” bond to form the advanced structure.

As used herein, the term “BY-001” refers to a composition ofhomomultimeric forms of bifunctional chimeric protein PD-L1/ Fc-gamma1at the ratio of 25% of dimer, 30% of tetramer and 45% of Hexamer.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response, suchas B cells, T cells, dendritic cells, natural killer cells, monocytesand macrophages, etc.

As used herein, the term “T cell” includes CD4+ Th1 and Th2 cells, CD3+and CD8+ cytotoxic T cells including self-reactive T cells, CD25+ andFOX3+ Treg cells and inducible Treg cells (iTreg).

As used herein, the term “B cells” refers to the B cell potential toproducing the antibodies including the self-reactive antibodies.

As used herein, the term “aggregated PD-L1” refers to the extracellularportion of PD-L1 of human or mouse origin at the aggregated statethereof.

As used herein, the term “aggregated Fc-gamma1” refers to the regions ofhinge, CH2 and CH3 of human IgG1 heavy chain of human or mouse origin atthe aggregated state thereof, in addition, any peptide combined to theregions of hinge, CH2 and CH3 of human IgG1 heavy chain of human ormouse origin at the aggregated state thereof is include.

As used herein, the term “inhibitory signal” refers to a signaltransmitted via an inhibitory receptor molecule on an immune cell. Suchas CTLA-4, PD-1 and Fc-gammaRIIB.

5.2. Mode for Invention

Hereinafter, the invention is explained in accordance with the examplesto further illustrate the application without limiting its scope.

In accordance with the object of the present invention, first structureof protein in one aspect, there is provided bifunctional chimericprotein in first structure of protein comprising a first functiondomain, the extracellular domain of PD-L1 protein or a fragment thereofand a second function domain, the human immunoglobulin Fc-gamma1 region.The bifunctional chimeric protein of current invention is stated as“PD-L1/Fc-gamma1 chimeric protein” or “PD-L1/Fc-gamma1 protein” or“PD-L1 chimeric protein” or “chimeric protein”. The extracellular domainof PD-L1 may be a polypeptide comprising an immunoglobulin V (V-set)like domain of PD-L1 and an immunoglobulin C (C-set) like domain ofPD-L1. The extracellular domain of PD-L1 is a domain exposed outside acell membrane, and may be a polypeptide comprising the amino acids atpositions 19 to 220 of sequence of FIG. 1B of current invention. Inaddition, the extracellular domain of PD-L1 or a fragment thereof may beof human origin. In addition, the extracellular domain of PD-L1 may haveabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homology to the polypeptide sequence consisting of the aminoacids at positions 19 to 220 of sequence of FIG. 1B. In addition, HumanIgG1 heavy chain constant domain (C region, 221˜449) of the sequence ofFIG. 1B is the region of Fc-gamma1.

The present invention provided, advanced structure of protein in anotheraspect, is bifunctional chimeric protein in advanced structure ofprotein comprising a composition of homomultimer of dimer, tetramer andhexamer of PD-L1/Fc-gamma1 chimeric protein (BY-001). All the Cysteinesin V-set and C-set and human immunoglobulin Fc-gamma1 region ofPD-L1/Fc-gamma1 thereof are kept in current invention for the —S—S—“disulfide” bond to form the advanced structure. Hence, once the PD-L1is fused to second domain components Fc-gamma1, it is a complexnaturally composed in dimer, tetramer and Hexamer, in fact PD-L1 dimer,tetramer and Hexamer are formed, on the other hand, Fc-gamma1 dimer,tetramer and Hexamer are formed also, for example as presented in FIG. 2of current invention.

To date, the subsets of immune cells of activated T cells, B cells anddendritic cells are related to the autoimmunity and rejection ofallografts, while monocytes/macrophages are one of the key subsetsrelated to inflammatory reaction, particularly to the chronicinflammation. It has highly recognized that the cell signaling pathwaysPD-1/PD-L1 and Fc-gammaRII/Fc-gamma are crucial in the regulation offunctions of the subsets of immune cells mentioned above.

Under physiological condition, PD-1 is expressed in activated T cellsand B cells and is bound and triggered by monomeric PD-L1, thereafter,the PD-1/PD-L1 pathway delivers the cell signaling of immune checkpoint.Thus PD-1/PD-L1 axis is one of the pathways that exert the key roles inthe establishing and maintenance of immune tolerance. The provided incurrent invention is the PD-L1 in chimeric protein at its aggregatedstate and is expected to act on the PD-1 signaling axis. To validate thefunction of PD-L1 at its aggregated forms as stated (FIG. 2) in currentinvention, in one embodiment as presented in FIG. 3 and FIG. 4 ofcurrent invention, the present invention provides validation that PD-1on immune cells is activated by the aggregated form of PD-L1 (BY-001),thereafter to deliver the cell signaling of immune checkpoint. Thebifunctional chimeric protein PD-L1/Fc-gamma1 comprising a first domainPD-L1 that binds to and triggers PD-1. For example, as shown in currentinvention, BY-001 inhibits the IL-2 production from the Jurkate cells(FIG. 3) and human PBMCs of healthy donor (FIG. 4) after the treatmentwith either OKT3 antibody or CKT3 antibody +CD28 antibody. OKT3 antibodyalong or together with CD28 antibody as the co-stimulator are commonlyused to trigger TCR signaling for the activation of CD3+ T cells. IL-2production is one of the biomarkers commonly to indicate T cellactivation. It has commonly believed that inflammatory reaction inautoimmunity or rejection of allografts are caused by inappropriateactivation of the self-reactive T cells and self-reactive B cells due tothe peripheral immune tolerance breakdown [1-3]). The current methods inthe treatment of autoimmune diseases or protection of allografts are tosuppress the activity of the mentioned self-reactive T cells andself-reactive B cells [5, 35]. To date, it has believed that cellsurface receptor of PD-1 is expressed only in activated T cells and Bcells [35]. The association between PD-1 and PD-L1 induces the activatedT anergy cell or B cell anergy or apoptosis, or alternatively theassociation induces the differentiation of Treg cell from Th cell [36].Therefore, the current invention provided homomultimeric forms ofPD-L1/Fc-gamma1 (BY-001), a novel concept of soluble chimeric proteins,is potential to treat certain immune and inflammatory disorders in onedimension.

According to the ability to activate or suppress the immune responseFc-gamma receptors are divided in two classes: hFc-gammaRI,hFc-gammaRIIA and hFc-gammaRIIIA are activating receptors via thecytoplasmic ITAM (immunoreceptor tyrosine-based activation motif),whereas hFc-gammaRIIB is suppressing receptor via the cytoplasmic ITIM(immunoreceptor tyrosine-based inhibitory motif) [37]. Fc-gammaRI is ahigh-affinity receptor that binds to only monomeric IgG and is expressedconstitutively in dendritic cells, monocytes and macrophages in bothhuman and mice[38]. In contrast to Fc-gammaRI, the other three typesexpress in most of the immune cell populations, except the B cells, andexhibit affinity to Fc-gamma of IgGs in antigen-antibody complex, amongthem Fc-gammaRIIB exerts the suppressive signaling through its bindingwith one Fc-gamma and crosslinking others Fc-gammaRIIA with othersFc-gamma in antigen-antibody complex. Furthermore, Fc-gammaRIIB isexpressed on B cell and appears to function in a B cell-autonomousmanner to regulate autoreactive cells in the periphery. There have beenextensively reported that Fc-gammaRII plays key role in autoimmunity andinflammatory disorders. Fc-gammaRII/Fc-gamma axis dominates theactivation and chemo-cytokines production profile of dendritic cells andmonocytes/macrophages, which is critical for the process ofinflammation, and also is the key role in maintenance of immunetolerance. The provided in current invention is the Fc-gamma1 inchimeric protein at its aggregated state and is expected to act on theFc-gammaRII signaling axis.

To validate the function of Fc-gamma1 at its aggregated forms as stated(FIG. 2) in current invention, in another embodiment, as a novel conceptof soluble chimeric proteins, presented in FIG. 5 and FIG. 6 of currentinvention, the present invention provides validation that Fc-gammaRIIBon immune cells is activated by the aggregated form of Fc-gamma1(BY-001), thereafter to deliver the suppressive cell signaling to immunecells. In current invention, the artificially aggregated formPD-L1/Fc-gamma1 (BY-001) in tetramer and hexamer are distinct from theFc form in Ag-Ab complex in structure according to diagram in FIG. 7 ofcurrent invention. As shown in current invention, for example, BY-001inhibits the IL-4 and TGF-betta production from the K562 cells and THP-1cells. It was reported that K562 cells express ortly Fc-gamma receptorII [39], while THP-1 cells express both Fc-gamma receptor I and II onthe cell surface[40]. Apparently, the activating receptor of Fc-gammaRIon THP-1 cell didn't influence the suppressive pathway ofFc-gammaRIIB/Fc-gamma1. The machinery of suppressive pathway maycomprise Fc-gamma1 bound with Fc-gammaRIIB and others Fc-gamma1 inmultimeric form crosslinked the Fc-gammaRIIA or RITC, thereafter, theinhibitory signaling is delivered and the activity of the cells aredecreased through the pathway.

Collectively, BY-001 exerts dual functions that in one hand binds to andtriggers the PD-1 pathway through multimeric PD-L1, and on the otherhand binds to Fc-gamma1RIIB through multimeric Fc-gamma1 to trigger thesuppressive signaling into the cells. Thereafter, BY-001 is capable toregulate the subsets of immune cells activated T cells and B cells,meanwhile to regulate the subsets of dendritic cell andmonocytes/macrophages, the immune cell populations tightly relate toautoimmunity and protection of allografts. Therefore, BY-001 ispotential to treat certain immune and inflammatory disorders. In thesetting of autoimmune, alloimmune and inflammatory diseases, thehomomultimeric forms of chimeric protein of this invention can reduceautoimmune, alloimmune and inflammatory manifestations by one or moremechanisms. For example, the PD-L1 extracellular domain inhomomultimeric forms of chimeric protein of this invention can bind tothe PD-1 on immune cell, such as an activated T cell or pre-B cell;while the second domain in homomultimeric forms of chimeric proteinbinds to and cross-links the FC receptors that express on dendritic celland macrophage and other immune cell bearing Fc-gammaRII or RIII, etc..Through these binding and cross-linking events, the receptors PD-1 forthe first domain trigger the T cell anergy, repress the B cellmaturation, differentiation of Treg cells and regulatory dendriticcells; the FC receptors for second domain of the chimeric protein mayinhibit the activated dendritic cell and macrophage in the inflammatorylocation. All these events are critical to induce and maintain theimmune tolerance of the body.

In addition, once the FC-gamma1 contained in chimeric protein anchoredto FC receptor on cell surface of dendritic cell and macrophage, nowmembrane-anchored, therefore, the chimeric protein of the presentinvention may mediate its activity by spanning two neighboring cells,and thereby establish an immune tolerant microenvironment atinflammatory site. Alternatively, for example, a PD-L1 containingchimeric protein can bind to the B7-1 costimulator on anantigen-presenting cell, thereby interfering with its costimulatory,immune-activating function.

As indicated in schematic diagram of FIG. 8, BY-001 is potential for thetreatment of autoimmune diseases and protection of allografts. Forexample, type 1 diabetes (T1D, Systemic Lupus Erythematosus (SLE),Rheumatoid Arthritis (RA) and inflammatory bowel disease (IBD)(including ulcerative colitis (UC) and Crohn's disease (CD)), multiplesclerosis (MS), scleroderma, and other diseases and disorders, such asPV (pemphigus vulgaris), psoriasis, atopic dermatitis, celiac disease,Chronic Obstructive Lung disease, Hashimoto's thyroiditis, Graves'disease (thyroid), Sjogren's syndrome, Guillain-Barre syndrome,Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis,primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis,polymyalgia rheumatica. Raynaud's phenomenon, temporal arteritis, giantcell arteritis, autoimmune hemolytic anemia, pernicious anemia,polyarteritis nodosa. Behcet's disease, primary bilary cirrhosis,uveitis, myocarditis, rheumatic fever, ankylosing spondylitis,glomerulenephritis, sarcoidosis, dermatomyositis, myasthenia gravis,polymyositis, alopecia areata, and vitilgo.

7. FIELD OF THE INVENTION

The present invention relates to the advanced structure of chimericprotein PD-L1/Fc-gamma1 composed of homomultimeric forms (BY-001),wherein the first domain, such as PD-L1, at the aggregated state bindsto and triggers the PD-1 and the second domain, such as Fc-gamma1, atthe aggregated state binds to and triggers Fc-gammal receptor type II.

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6.1. An active composition (BY-001) of homomultimeric forms of chimericprotein PD-L1/Fc-gamma1 comprising the forms of dimer, tetramer andHexamer, wherein the probable ratio of the forms is at 25% of dimer, 30%of tetramer and 45% of hexamer respectively (FIG. 2). 6.2. A form ofhomomultimeric chimeric protein PD-L1/Fc-gamma1 in advanced structureconsisting of multiple monomer of chimeric protein PD-L1/ Fc-gamma1,wherein the monomer of chimeric protein PD-L1/Fc-gamma1 contains twodistinct function domains. 6.3. The two distinct function domains in themonomeric chimeric protein PD-L1/ Fc-gamma1 according to claim 2consisting of the PD-L1 domain (extracellular domain of human PD-L1) andthe Fc-gamma1 domain (region of human IgG1 Fc), wherein the PD-L1 domaincontains the portions of immunoglobulin V like region (V-set region,19˜127 amino-acids, exon-2) and immunoglobulin constant like region(C-set region, 128˜220 amino-acids, exon 3), and the Fc-gamma1 domaincontains the regions of hinge, CH2 and CH3 in human IgG1 heavy chain(FIG. 1). 6.4. BY-001 (a composition of bifunctional homomultimericforms of chimeric protein PD-L1/Fc-gamma1) according to claims 1 and 3comprising the polypeptide PD-L1, wherein the aggregated form of PD-L1portion bind to and trigger PD-1 (CD279) expressed on T cells. 6.5.BY-001 (a composition of bifunctional homomultimeric forms of chimericprotein PD-L1/Fc-gamma1) according to claim1 and 3 comprising thepolypeptide Fc-gamma1, wherein the aggregated form of Fc-gamma1 portionbind to Fc-gammaRIIA/B and trigger the suppressive function ofFc-gammaRII expressed on monocyte/macrophages or progenitor oferythrocyte. 6.6. A method of treating CD3+ CD28+ immune cellscomprising administering effective amount of BY-001 according to claim4, wherein aggregated PD-L1 portion of BY-001 suppresses the activity ofT cell (FIG. 3). 6.7. A method of treating human peripheral bloodmonocyte (hPBMC) comprising administering effective amount of BY-001according to claim 4, wherein aggregated PD-L1 portion of BY-001suppresses the activation of PD-1 bearing lymphocytes among the PBMC(FIG. 4). 6.8. A method of treating immune cells bearing Fc-gammaRIIonly comprising administering effective amount of BY-001 according toclaim 4, wherein the homomultimeric Fc-gamma1 of BY-001 bind to andtrigger the Fc-gammaRIIA/B on immune cells to suppress the cytokinesproduction (FIG. 5). 6.9. A method of treating immune cells comprisingimmune cells bearing Fc-gammaRI and Fc-gammaRII administering effectiveamount of BY-001 according to claim 4, wherein the homomultimericFc-gamma1 of BY-001 bind to and trigger the Fc-gammaRIIA/B on immunecells to suppress the cytokines production (FIG. 6). 6.10. Thebifunctional proof of the domains contained in BY-001 according toclaims 6, 7, 8 and 9 comprising the features of triggering the pathwaysof PD-1/PD-L1 and Fc-gamma1RIIB/Fc-gamma, wherein the features inheritthe characteristics in vitro and in vivo that have been published inhighly recognized scientific journals as mention in the part ofbackground. 6.11. BY-001 according to claim 10 comprising apharmaceutical composition for the treatment of immune diseases orprotection of allografts, wherein the therapeutic potential of BY-001 isindicated